• alveolar model
• in silico-in vitro approach
• 3D spherical membrane
• multiscale fluidic system
• breathing
• magneto-responsive hydrogels

The increasing incidence of respiratory disorders in recent decades has encouraged investigations into human pulmonary pathophysiology to support human health. Despite scientific improvements, current technologies fail to meet all the constitutive elements of the lung’s functional units, i.e., the alveoli. From a technological viewpoint, the existing challenges concern the physiological reproduction of the alveolar curvature, the mechanical properties of the pulmonary microenvironment, and breathing actuation mechanisms.
My PhD research aimed at developing alveolar models through integrated in silico-in vitro strategies to recapitulate the main dynamic and mechano-architectural features of the alveolar barrier. The alveolar model realised can mimic the pulmonary parenchyma's mechanical properties, the alveolar three-dimensional spherical architecture with the ALI (ALI), the physiological air and blood dynamics, and model actuation or “breathing” through traditional and more physiological methods.
First, an in silico model was designed and implemented to identify the ideal material combination capable of providing a substrate with a mechanical response within the range of the lung parenchyma. The material combinations -consisting of alginate, gelatin, and agarose - were subsequently employed to replicate the 3D spherical architecture; more specifically, two approaches were studied. The first included developing a combined in silico-in vitro method that allowed fabricating alginate-based core-shell spheroids with controlled geometry. Since they can host different cellular populations, core-shell spheroids successfully mimic the liquid-liquid interface and offer a viable technique for fabricating 3D complex biological environments. The main challenges with this technology regard the reproducibility of the air-core spheroids for mimicking the ALI and the integration of the spheroids inside a fluid-dynamic framework.
For these reasons, during my studies, I proposed a second technique based on the design of 3D spherical agarose membranes. The membranes were manufactured via moulding techniques and a sequence of temperature-guided and enzymatic crosslinking, drying and hydration processes. Moreover, the fabrication procedure was extended for fabricating downscaled membranes towards alveolar diameters to provide cells with an alveolar-like curvature. The membranes were characterised in terms of mechanical, functional, and biological features, yielding excellent results in replicating the corresponding qualities of the alveolar barrier. To facilitate the characterisation of permeability and cell-adhesive properties, a cell culture prototype was designed to adapt the membranes to Transwell-like inserts.
Using the prototype, the membranes could be easily integrated into a two-compartmental fluidic platform. Therefore, the next step concerned the design of the fluidic systems that could incorporate the membranes for modelling the alveolar sac while preserving the physiological ratio between the diameters of the alveolar duct and the alveoli. In silico models of liquid and air flows were established to find the optimal inlet velocity conditions and architecture while respecting physiological shear stress, heartbeat and breathing frequency, and flow field topology. The fluidic platforms were produced employing stereolithographic processes and a transparent biocompatible resin. Furthermore, a PDMS insert specifically designed for housing membranes was developed to assist in the membrane docking and fluidic system sealing.
The replication of breathing was the final challenge of the PhD study; two methods were investigated. The first was the development of an in silico-in vitro approach for determining the ideal pressurised air values that can provide membrane deformation within the physiological range of the alveolar barrier. The computational findings were validated by measuring the deformation of the membrane as a function of the inflow pressure. Although pressurised air can provide the actuation required to replicate breathing, this method resembles forced ventilation.
As a result, in the final phase of my PhD study, a more physiological approach based on magneto-responsive agarose gels was designed to mimic better the actuation provided by the respiratory muscles during breathing movements. An in silico-in vitro strategy was developed to identify the optimal combination of ferrite nanoparticles and agarose concentration capable of providing hydrogel deformation within the physiological range due to the application of an external magnetic field. Given the promising results, an in silico study was conducted to assess the feasibility of embedding the agarose magneto-responsive gel into the alveolar sac in vitro model to provide the conditions for more physiological membrane actuation.
The proposed multiscale alveoli in vitro model presented in this doctoral project has shown promising throughputs, highlighting the potential of in vitro models for investigating the impact of inhaled molecules on human alveoli in pathophysiological scenarios. In conclusion, this study contributes to bridging the existing gap between the “proof-of-concept” use of lung in vitro models and their subsequent preclinical and clinical applications.